The ability to direct differentiation of stem cells is crucial for more direct mimicry of tissue development processes, and it is particularly important to advance stem cell-based approaches to tissue regeneration. Existing strategies to direct stem cell differentiation focus primarily on supplementing culture environments with protein growth factors, which are capable of inducing differentiation down specific lineages. An emerging approach in regenerative medicine involves transfecting cells with genes that encode inductive growth factors, leading to transgenic production of these factors by cells within a growing tissue. Previous studies demonstrate that gene delivery can be used to induce generation of several tissue types. However, previous approaches have not yet achieved a high level of spatial control over growth factor production, which is likely to be vital for regeneration of complex multicellular tissues such as vascular tissue. This proposal describes a research program that aims to achieve highly controlled transfection of mesenchymal stem cells (MSCs) with plasmid DNA encoding inductive growth factors. In particular, we plan to exploit well-characterized complementary DNA interactions to controllably sequester plasmid DNA on hydrogel substrates. We hypothesize that DNA immobilized to a substrate via complementary DNA interactions can be used to transfect cells that are adhered to the same substrate, and that spatial patterning of the immobilized DNA will enable spatial control over transfection. We specifically aim to: 1) develop and characterize hydrogels that simultaneously present peptide ligands for cell adhesion and oligonucleotide handles for localized plasmid DNA sequestering; 2) examine the dependence of transfection efficiency on plasmid DNA availability and cell adhesion characteristics; and 3) achieve spatially patterned transfection of hMSCs with genes encoding two key inductive growth factors: vascular endothelial growth factor and platelet-derived growth factor. These factors are chosen based on their ability direct differentiation of MSCs into endothelial and smooth muscle cells, respectively. We expect that the proposed research will initially produce a more complete understanding of substrate-enhanced gene delivery as well as an enabling technology for vascular tissue regeneration. Ultimately, our gene delivery approach for directed stem cell differentiation can, in principle, be applied to any adherent stem cell type and any growth factor. [unreadable] [unreadable] [unreadable] [unreadable]